Biochemical Characterisation of The Plasmodium falciparum. Chloroquine Resistance Transporter

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Biochemical Characterisation of The Plasmodium falciparum Chloroquine Resistance Transporter

Fadi Baakdah

Institute of Parasitology McGill University, Montreal

August 2020

A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Doctor of Philosophy

© Fadi Baakdah

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ABSTRACT

The emergence of resistance to commonly used antimalarials significantly hindered global efforts in eliminating malaria and cost the human race losses of lives in millions. Plasmodium falciparum parasites are the most accountable for morbidity and mortality compared to the other species that infect humans. At the present time, artemisinin combination therapies is the approach used in the field to treat malaria infected people and has shown tremendous success. However, resistance to these combinations recently emerged and the pattern of progression and spreading is alarming. Chloroquine once was the first-line drug for treatment of malaria infected people, however, it became in-effective due to the spread of chloroquine resistant strains. Many attributes of chloroquine, at the time when it was effective, were desired and as such the field took on different approaches to revive it. Some people took an approach to withdraw the use of chloroquine for a significant period of time resulting in the emergence of chloroquine sensitive strains. Others looked into modifying the structure of chloroquine in order to make derivatives that would be an improvement on the original. Additionally, others went on to investigate the molecular mechanism by which the parasite confers resistance to chloroquine. Presently, it is well known that mutations in the chloroquine resistance transporter (PfCRT), expressed on the membrane of a lysosome-like organelle in the parasite, the digestive vacuole (DV), are the primary determinants of chloroquine resistance. The physiological role and normal substrates are still matters of speculation but the protein seems to be important for the parasite survival because knockout-PfCRT clones could not be established. The crystal structure was resolved showing the spatial arrangement of the polypeptide chain relative to the juxtaposition of the transmembrane domains forming the central cavity where drugs would interact with PfCRT.

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II Given PfCRT’s role in chloroquine resistance, we thought if chloroquine was slightly modified it would bypass PfCRT resistance mechanism. The first experimental manuscript thesis, we examined the antimalarial activity of 16 novel chloroquine derivatives against chloroquine- sensitive and -resistant Plasmodium falciparum strains. Only two compounds (e.g., AQ-13 and AQ-129) showed effects that surpassed chloroquine’s effect on chloroquine resistant strains that were examined previously but not to the extent of their relationship with PfCRT. Our results demonstrate that AQ-13 and AQ-129 are poor substrates of PfCRT and thus more effective against chloroquine resistant parasites.

In the 2^nd manuscript, we describe the high resolution characterisation of an antiserum raised against the full-length C-terminal domain of PfCRT. An IgG pool that recognises a de- phosphorylated Ser411 epitope was extracted and used as a tool to monitor the phosphorylation status of residue Ser411. This pool of IgG`s identified the presence of an Ser411 de- phosphorylated homodimer form of PfCRT that does not localise to the DV membrane as does the monomer PfCRT. We also show that PfCRT monomer in chloroquine-sensitive strain (3D7) is significantly more phosphorylated than in chloroquine-resistant strain (Dd2-H) at Ser411, suggesting a possible functional role for this residue in drug resistance.

In the last manuscript, we describe the adoption of mammalian HEK-293F cells as a heterologous system to study PfCRT function. Using HEK-293F cells stably expressing PfCRT wild-type and mutants, we show mutant-PfCRT to cause a significant acidification of the lysosomes, relative to wild-type PfCRT. We also provide direct evidence that acidification was mediated through mutant-PfCRT, since using a proline-165-modified mutant-PfCRT clone restored the acidification

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III of lysosomes to wild-type PfCRT levels. Thus, results of this study show for the first time the role of Pro165 in mutant-PfCRT function.

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IV

ABRÉGÉ

L'émergence d'une résistance aux antipaludiques couramment utilisés a considérablement entravé les efforts mondiaux visant à éliminer le paludisme et a coûté des millions de vies à la race humaine. Les parasites de l’espèce Plasmodium falciparum sont les plus responsables de la morbidité et de la mortalité par rapport aux autres espèces qui infectent l’humain. À l'heure actuelle, les thérapies combinées à l'artémisinine sont l'approche utilisée sur le terrain pour traiter les personnes infectées par le paludisme et ont connu un énorme succès. Cependant, une résistance à ces combinaisons a récemment émergé et le schéma de progression et de propagation est alarmant. La chloroquine était autrefois le médicament de première intention pour le traitement des personnes infectées par le paludisme, mais elle est devenue inefficace en raison de la propagation de souches résistantes à la chloroquine. De nombreux attributs de la chloroquine, au moment où elle était efficace, étaient souhaités et, à ce titre, la science a adopté différentes approches pour la faire revivre. Certaines personnes ont adopté l’approche de retirer l'utilisation de la chloroquine sur le terrain pendant un bon bout de temps, entraînant l'émergence de souches sensibles à la chloroquine. D'autres ont envisagé de modifier la structure de la chloroquine afin de fabriquer des dérivés qui seraient une amélioration par rapport à l'original. De plus, d'autres ont poursuivi leurs recherches sur le mécanisme moléculaire par lequel le parasite confère une résistance à la chloroquine. Actuellement, il est bien connu que les mutations du transporteur de résistance à la chloroquine (PfCRT), exprimées sur la membrane d'un organite de type lysosome chez le parasite, la vacuole digestive, sont les principaux déterminants de la résistance à la chloroquine. Le rôle physiologique et les substrats normaux de cette protéine sont encore des sujets de spéculation mais la protéine semble être importante

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V pour la survie du parasite car les clones de Plasmodium déficient en PfCRT n'ont pas pu être établis. La structure cristalline de la protéine PfCRT a été résolue montrant l'arrangement spatial de la chaîne polypeptidique par rapport à la juxtaposition des domaines transmembranaires formant la cavité centrale où les médicaments interagiraient avec PfCRT.

Étant donné le rôle des protéines PfCRT dans la résistance à la chloroquine, nous pensions que si la chloroquine était légèrement modifiée, elle contournerait le mécanisme de résistance à la protéine PfCRT. Dans la première partie de cette thèse, nous avons examiné l'activité antipaludique de 16 nouveaux dérivés de la chloroquine contre les souches de Plasmodium falciparum sensibles et résistantes à la chloroquine. Seuls deux composés (par exemple, AQ-13

et AQ-129) ont montré des effets supérieurs à l'effet de la chloroquine sur les souches résistantes à la chloroquine qui ont été examinés, mais pas dans la mesure de leur relation avec PfCRT. Nos résultats démontrent que l'AQ-13 et l'AQ-129 sont de mauvais substrats de la protéine PfCRT et donc plus efficaces contre les parasites résistants à la chloroquine.

Dans le chapitre 3, nous avons décrit la caractérisation haute résolution d'un antisérum dirigé contre le domaine entier C-terminal de PfCRT. Un ensemble d'IgG qui reconnaît un épitope Ser411 déphosphorylé a été extrait et utilisé comme outil pour surveiller l'état de phosphorylation du résidu Ser411. Cet ensemble d'IgG a identifié la présence d'une forme homo- dimère Ser411 déphosphorylé de PfCRT qui ne se localise pas à la membrane de la vacuole digestive comme le fait le monomère PfCRT. Nous avons montré également que le monomère PfCRT dans la souche sensible à la chloroquine (3D7) est significativement plus phosphorylé que dans la souche résistante à la chloroquine (Dd2-H) à la position Ser411, suggérant un rôle fonctionnel possible pour ce résidu dans la résistance aux médicaments.

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VI Dans le chapitre 4, nous avons décrit l’utilisation des cellules HEK-293F de mammifères comme système hétérologue pour étudier la fonction de PfCRT. En utilisant des cellules HEK-293F exprimant de manière stable les protéines PfCRT originale et mutante, nous avons montré que la protéine PfCRT mutante provoque une acidification significative des lysosomes, par rapport à la protéine PfCRT originale. De manière très significative, nous avons fournis également des preuves directes que l'acidification a été médiée par la protéine PfCRT mutante, car l'utilisation d'un clone mutant-PfCRT qui a été modifié par la proline 165 a restauré l'acidification des lysosomes au même niveau que celui de la protéine PfCRT originale. Ainsi, les résultats de cette étude montrent pour la première fois le rôle du résidu Pro165 dans la fonction de la protéine mutante-PfCRT.

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VII

ACKNOWLEDGEMENTS

Firstly, I would like to thank my supervisor Dr. Elias Georges. Thank you for taking me into your lab and giving me the opportunity to do my PhD. It is thanks to you that I have had the chance to develop my skills as a researcher. Thank you for being very generous with your time and your enthusiasm for my project. You have been a great supervisor to me and I am very grateful.

Past and present lab members, we have been through a lot over the years, you all were an important part of my journey. We had a lot of laughs, we shared science and we were there for each other when we needed. You guys were simply the perfect lab family members to have: Dr.

Sonia Edaye, Georgia Limniatis, Rowa Bakhadlag, Zahra Sahaf, Kristin Lee and Haritha Menon

A special thanks for my good friend Georgia and her whole family who have stood by me and my family through difficult times. You guys were amazing and I will never forget that. In the same sense I would like to thank Dr. Anwer Hasil and his family, Dr. Najmeh Nikpour, Dr. Manjurul Haque, Dr. Tim Geary and Dr. Robin Beech. Truly all of your efforts helped me get this far.

I’d like to thank my good friend Sami Lakhmiri who was like a brother to me in Montréal. Also for his kind parents, their support and many invitations for breakfast and dinner. Your mom knows how to throw a party!

A thank you to the malaria guys Jeffry Agyapong, Shararreh Maleki and Tosin Opadokun for great talks and fun times.

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VIII A thank you to my wife and my daughter who were my back bone throughout my stay in Montréal and tolerating this whole experience with me. Both of our projects had a lot of crazy events but we always had each other’s backs.

A special thank you to my parents and my whole family for all their support and encouragement and grateful for all that they had done to help me reach my goals.

The Covid-19 pandemic did not make things easy but many people in their positions made life better in such difficult times. I’d like to thank you for all what you had done and all of your help and support and courage.

Many thanks to anyone who supported me if I forgot to mention them in and out of the institute of parasitology.

Lastly, I thank my sponsors: King Abdulaziz University for covering my tuition at McGill University and stay in Canada in coordination with the Saudi Arabian Cultural Bureau-Ottawa (SACB).

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CONTRIBUTION OF AUTHORS

The experimental work presented in this thesis was designed, executed and performed by the author under the supervision of Dr. Elias Georges, who was involved in experimental design, data presentation and editing of this thesis and the manuscripts included.

In the first manuscript (chapter 2), Benita Kapuku synthesized the chloroquine derivatives and Julia Hageman worked with Benita Kapuku only on the synthesis of compounds AQ-13 and AQ- 129 used in the study under the supervision of Dr. David Scott Bohle from the Department of Chemistry, McGill University.

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CONTRIBUTION TO ORIGINAL KNOWLEDGE

The following aspects described in this thesis are considered original contributions to our knowledge of malaria drug resistance.

MANUSCRIPT I

Fadi Baakdah, Benita Kapuku, Julia Hageman, David Scott Bohle and Elias Georges.

Characterization of 4-Aminoquinoline Derivatives against Chloroquine Resistant Strains of Plasmodium falciparum. (Manuscript in preparation).

This manuscript examined 16 novel chloroquine derivatives modified at 3^rd position of the quinoline moiety/ the side-chain of chloroquine that included two previously demonstrated effective compounds on chloroquine resistant strains, AQ-13 and AQ-129. The study found that AQ-13 and AQ-129 were the most effective against chloroquine resistant strains and showed for the first time that they are poor substrates of PfCRT using PfCRT isogenic clones. Selecting for resistant mutants to AQ-13 and AQ-129 for a period of 3 months was not established suggesting that resistance is not an easy task to achieve to these compounds by the parasites. We also demonstrate AQ-13 and AQ-129 inhibit beta-hematin formation in-vitro as in the case of chloroquine. Taken together, this work demonstrates that AQ-13 and AQ-129 are upgrade chloroquine derivatives better than original chloroquine.

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MANUSCRIPT II

Fadi Baakdah and Elias Georges.

High resolution mapping of PfCRT antiserum identifies a phosphorylated PfCRT at Ser411 in the parasite digestive vacuole. (Manuscript in preparation).

Resistance to chloroquine via mutant-PfCRT was shown to involve phosphorylation of Ser33 on the N-terminal of PfCRT. Here we extracted and characterized an anti-Ser411 pool of IgG`s from a PfCRT antibody raised against synthetic de-phosphorylated PfCRT C-terminal peptide. This special pool of IgG`s showed the presence of homodimer-PfCRT not localized to the DV membrane and is de-phosphorylated on Ser411 in contrast with the monomer form. It also showed that chloroquine sensitive strain 3D7 is significantly more phosphorylated than chloroquine resistant strain Dd2-H at Ser411 suggesting a possible functional role. Taken together, we have a novel tool to monitor the status of residue Ser411 in PfCRT.

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MANUSCRIPT III

Fadi Baakdah and Elias Georges.

Substitution of Pro165 in transmembrane 4 of chloroquine resistance transporter PfCRT abolishes lysosome acidification function in stably transfected HEK-293F Cells. (Manuscript in preparation).

Studying PfCRT in the parasite is challenging. Therefore, we stably expressed wt-PfCRTÊTSE and mut-PfCRT^NKAQ in HEK-293F cells and selected clones for each one. We show that the PfCRT protein in all the clones localised to the lysosomal membrane. Mut-PfCRT^NKAQ clones demonstrated significant acidification relative to wt-PfCRTÊTSE clones measured by accumulation of pH sensitive dyes Acridine orange and LysoOrange Indicator reagent. We also demonstrate the abolishment of the acidification function mediated by mut-PfCRT^NKAQusing our proline mutated clone mut-P165A-PfCRTÊTSE. This showed that acidification was directly mediated through mutant PfCRT. Moreover, we demonstrate an essential role for Pro165 in mutant PfCRT lysosomal acidification function.

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LIST OF ABBREVIATIONS

ABC ATP binding cassette

ABCG2 ATP binding cassette member G2

ACT Artemisinin based Combination Therapies

ART Artemisinin

ATP Adenosine Triphosphate

BCRP Breast Cancer Resistance Protein

CQ Chloroquine

CQR Chloroquine Resistance

CQS Chloroquine Sensitive

DAPI 4’,6-diamidino-2-phenylindole

DDT DichloroDiphenylTrichloroethane

DHFR Dihydrofolate reductase

DHPS Dihydropteroate synthase

DMSO Dimethylsulfoxide

DNA Desoxyribonucleotide adenosine

DV Digestive Vacuole

EDTA Ethylenediaminetetraacetic acid

GSH Glutathione

GFP Green fluorescent protein

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HF Halofantrine

IC50 Inhibitory concentration for 50%

kDa Kilo Dalton

MDR1 Multi Drug Resistance 1

MQ Mefloquine

MW Molecular weight

NBD Nucleotide Binding Domain

PBS

P. falciparum

Phosphate Buffer Saline Plasmodium falciparum

PfCRT Plasmodium falciparum Chloroquine Resistance Transporter PfMDR1 Plasmodium falciparum Multi Drug Resistance 1

PfMRP1 Plasmodium falciparum Multidrug Resistance Protein 1 PfPNP Plasmodium falciparum cytosolic purine nucleoside

phosphorylase

Pfkelch13 Plasmodium falciparum Kelch propeller protein 13

Pgp Pglycoprotein

QN Quinine

RBC Red blood cell

TM Transmembrane

TMD Transmembrane Domain

VP Verapamil

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WHO World Health Organization

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LIST OF FIGURES CHAPTER 1.

Literature review

Figure 1. Malaria global distribution map ... 6

Figure 2. The Life-cycle of the human malaria parasite ... 7

CHAPTER 2. Characterization of 4-Aminoquinoline Derivatives against Chloroquine Resistant Strains of Plasmodium falciparum

Figure 1. Group A CQ-derivatives. ... 49

Figure 1. Group B CQ-derivatives. ... 50

Figure 2. Anti-Plasmodial activity of Group A CQ-analogs. ... 51

Figure 3. Anti-Plasmodial activity of Group B CQ-analogs (part1). ... 53

Figure 3. Anti-Plasmodial activity of Group B CQ-analogs (part2). ... 54

Figure 4. Effects of AQ-13 and AQ-129 on proliferation of CQS and CQR strains (3D7 and Dd2-H) of P. falciparum without/with VP. ... 56

Figure 5. Effects of AQ-13 and AQ-129 on proliferation on additional CQS (HB3, D10) and CQR (7G8 and K1) strains of P. falciparum without/with VP. ... 58

Figure 6. Effects of AQ-13 and AQ-129 on proliferation on isogenic clones C2^GCO3 and C4^Dd2 of P. falciparum without/with VP. ... 60

CHAPTER 3. High resolution mapping of PfCRT antiserum identifies a phosphorylated PfCRT at Ser411 in the parasite digestive vacuole

Figure 1. Testing the specificity of anti-C PfCRT. ... 73

Figure 2. Testing anti-Ser411 PfCRT specificity. ... 83

Figure 3. Reactivity of pool2 IgG`s to PfCRT in chloroquine sensitive 3D7 and chloroquine resistant Dd2-H. ... 85

Figure 4. IFA on 3D7 parasites: anti-C (A, B, C and D) [15] and pool 2 IgG`s (E, F, G and H). ... 87

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CHAPTER 4. Substitution of Pro165 in transmembrane 4 of chloroquine resistance transporter PfCRT abolishes lysosome acidification function in stably transfected HEK-293F Cells

Figure 1. Sequence alignment of wild-type and mutant PfCRT amino acid sequences. ... 100 Figure 2. Stable expression of wild-type and mutant-PfCRT in HEK-293F cells. ... 101 Figure 3. Localisation of PfCRT in HEK-293F cells. ... 103 Figure 4. Accumulation of AO and LO in HEK-293F cells stably expressing wild-type and mutant- PfCRT. ... 104 Figure 5. PfCRT homology modeling with P-165-A mutation in PfCRT. ... 108

LIST OF TABLES Chapter 2.

Characterization of 4-Aminoquinoline Derivatives against Chloroquine Resistant Strains of Plasmodium falciparum

Table 1. Summary of screened compounds IC50 values. ... 55 Table 2. Summary of AQ-13 and AQ-129 effects on the proliferation of CQS and CQR strains (3D7 and Dd2-H) of P. falciparum without/with VP. ... 57 Table 3. Summary of AQ-13 and AQ-129 effects on the proliferation on additional CQS (HB3, D10) and CQR (7G8 and K1) strains of P. falciparum without/with VP. ... 59 Table 4. Summary of AQ-13 and AQ-129 effects on the proliferation on isogenic clones C2^GCO3 and C4^Dd2 of P. falciparum without/with VP. ... 61

CHAPTER 4. Substitution of Pro165 in transmembrane 4 of chloroquine resistance transporter PfCRT abolishes lysosome acidification function in stably transfected HEK-293F Cells

Table 1. Primers and reaction conditions for mut-PfCRT^ETSE. ... 111 Table 2. Primers and reaction conditions for mut-P165A-PfCRT^ETSE. ... 111

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GENERAL INTRODUCTION

Malaria is a blood-borne disease that shortened the life-span of millions of people especially in endemic regions. Plasmodium parasites are of many species, however, only a few cause the human malaria disease with the falciparum species being the most problematic. To survive in humans, the parasites made the host red blood cells their normal living space where they evolve through different life-stages of their erythrocytic cycle. Malaria treatment requires the use of established antimalarial drugs that exert their effects on different targets in the parasite. Based on their chemical structure and mode of action these compounds can be divided into five groups:

quinolines, antifolates, antimicrobials, hydroxynaphthaquinones and artemisnins [1, 2].

Quinoline based drugs are an important group of antimalarials that possess the heterocyclic aromatic quinoline ring. Quinine was the first antimalarial that was used for treatment replaced later by its derivative chloroquine [3]. Chloroquine, was the first-line antimalarial drug to treat malaria -infected individuals by virtue of its efficacy, safety and cost of synthesis and affordability until the parasites formulated a mechanism to resist its toxic effects [4]. Currently, it is recommended by the WHO that artemisinin combination therapies or ACTs are to be used for treatment as first-line which showed great success in alleviating the malaria burden and saved many lives [5]. However, resistance has emerged already to these combinations starting from Cambodia in the east and has now traveled west to the African continent [6-8]. Drug resistance is a continuous problem in the malaria field and the only means of defence against these pathogens is either blocking transmission of the parasite by controlling the distance between people and the female Anopheles mosquito vector using insecticidal bed nets or the use of effective antimalaria drugs. Additionally, an effective vaccine is still not ready to be used in the

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2 field yet [9]. As limited as our arsenal to take on malaria may be, the antimalaria drugs still remain our best means to treat and prevent malaria infections and the field is always exploring new compounds that can tackle current and future resistance mechanisms. Investigating the molecular resistance mechanisms is crucial to our understanding of molecular drug targets to design better drugs. It is well established that mutations in PfCRT are the primary determinants of chloroquine resistance [10]. The physiological substrate(s) as well as the native role of this transporter are still matters of speculation. The crystal structure was recently resolved but left us with questions about how this protein dynamically moves and function [11].

Therefore, the main objective of this thesis was to biochemically characterize PfCRT and its interactions with novel chloroquine derivatives. This will be illustrated in this thesis in an organised manner. Briefly, Chapter one describes the current knowledge of malaria drug resistance, antimalaria drugs used in the field and the different proteins involved in such mechanisms. Chapter 2 addresses the antimalarial activity of novel chloroquine derivatives and their relationship to PfCRT. Chapter 3 describes the high resolution mapping and characterisation of antibodies raised against PfCRT C-terminal domain and testing their ability to recognise a specific de-phosphorylated epitope to PfCRT at serine-411 that suggests to having a functional role. Chapter 4 addresses the possible functions PfCRT may have in the parasite by stably expressing PfCRT, wild-type and mutant forms, heterologously in HEK-293F cells. In addition to exploring the functional role of pro165 residue in transmembrane domain 4.

References

1. Müller, I.B. and J.E. Hyde, Antimalarial drugs: modes of action and mechanisms of parasite resistance. Future microbiology, 2010. 5(12): p. 1857-1873.

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3 2. Travassos, M. and M.K. Laufer, Antimalarial drugs: An overview. UpToDate Waltham MA.(ultimo

acceso 26 dic 2014), 2012.

3. Achan, J., et al., Quinine, an old anti-malarial drug in a modern world: role in the treatment of malaria. Malaria journal, 2011. 10(1): p. 144.

4. Hay, S.I., et al., The global distribution and population at risk of malaria: past, present, and future.

The Lancet infectious diseases, 2004. 4(6): p. 327-336.

5. Nosten, F. and N.J. White, Artemisinin-based combination treatment of falciparum malaria. The American journal of tropical medicine and hygiene, 2007. 77(6_Suppl): p. 181-192.

6. Witkowski, B., et al., Reduced artemisinin susceptibility of Plasmodium falciparum ring stages in western Cambodia. Antimicrobial agents and chemotherapy, 2013. 57(2): p. 914-923.

7. Lu, F., et al., Emergence of indigenous artemisinin-resistant Plasmodium falciparum in Africa. New England Journal of Medicine, 2017. 376(10): p. 991-993.

8. Roper, C., et al., Molecular surveillance for artemisinin resistance in Africa. The Lancet Infectious Diseases, 2014. 14(8): p. 668-670.

9. Aaby, P., et al., WHO’s rollout of malaria vaccine in Africa: can safety questions be answered after only 24 months? BMJ, 2020. 368.

10. Fidock, D.A., et al., Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Molecular cell, 2000. 6(4): p. 861-871.

11. Kim, J., et al., Structure and drug resistance of the Plasmodium falciparum transporter PfCRT.

Nature, 2019. 576(7786): p. 315-320.

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Chapter 1 LITERATURE REVIEW

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1. History and prevalence

Malaria is a blood borne human disease threatening the lives of millions of people, most of whom are children under five years of age. Periodic fever and chills are symptoms that reveal the onset of infection with malaria. These symptoms have been documented by the early Chinese approximately 2700BC and in early Greek writings, evidence showing that it is a very old disease that still is prevalent today [1, 2]. The name malaria stems from the Italian term mal` aria, which literally translates to bad air because it was thought that swamp fumes caused the illness. It wasn’t until French physician, Alphonse Lavern, observed crescent-like bodies in the blood of an infected soldier seen for the first time under light microscopy in the 18^th century [2]. These bodies were later designated as parasites of the Plasmodium species. To date, there are five species that infect humans: Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium falciparum (P. falciparum) and Plasmodium knowlesi (which infects macaque monkeys but has now been established as a zoonotic human pathogen as well [3]). Indeed, the most fatal species of Plasmodium that causes human disease is the falciparum species. This parasite species is responsible for approximately 90% of the total malaria cases in the African region. The latest World Malaria Report of 2018 estimated 216 million infections and ~445,000 deaths (figure 1) [4]. The most affected parts of the world are African countries, India, Southeast Asian and Latin American countries. North American countries and Europe do have some malaria cases;

however, these cases are now called “airport malaria” because they are due to immigration from traveling through endemic countries with malaria [5].

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Figure 1. Malaria global distribution map (adopted from World Malaria Report 2018 [4]).

2. Life-cycle of P. falciparum

The eukaryotic intracellular protozoan P. falciparum parasite is transmitted mainly through the bite of an infected female Anopheles mosquito species. This route of transmission was discovered by Ronald Ross who demonstrated the cycle by allowing mosquitoes to feed on infected patients with malaria [6]. The life-cycle involves three stages between the human host and the mosquito vector. When a female Anopheles mosquito takes a blood meal from an infected person, gametocytes start their sexual cycle by forming ookinetes that eventually develop into oocysts.

The oocysts produce over 10,000 sporozoites that migrate to the salivary glands of the mosquito.

The injection of the sporozoites into the human host marks the beginning of the exoerythrocytic stage. The sporozoites migrate to the liver and develop into schizonts. Later, merozoites burst

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7 out of these schizonts into the blood stream. When merozoites infect a red blood cell, it marks the start of the erythrocytic phase.

Figure 2. The Life-cycle of the human malaria parasite (adopted from Klein 2013 [7]).

The first stage is the ring stage, characterised by a chromatin dot with a light cytoplasm. Rings mature to trophozoites that have a distinctive important organelle, the DV, where hemoglobin metabolism occurs, resulting in the formation of the malaria pigment hemozoin, which is visible with light microscopy. Later in the life cycle, trophozoites develop into schizonts that generate multiple merozoites. The merozoites are then released and invade other host red blood cells (RBCs). However, not all merozoites that invade the host RBCs go from rings to schizonts; some

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8 develop into sexual stages, macro- and micro- gametocytes in a process called gametocytogenesis. This stage, when taken up by a female Anopheles mosquito continues the sexual life cycle [8].

3. The malaria human disease and its clinical manifestations

Proper laboratory diagnosis of infected individuals, especially the most vulnerable as children under five years of age, pregnant women and the immunocompromised, is key to accelerating their treatment and recovery. Suspected cases of malaria are usually diagnosed via blood tested in two different ways: a) under light microscopy examination by screening for the presence of parasites and estimating the intensity of infection and, b) a rapid diagnostic test (RDT) which is an antigen based detection system detecting antigens for Plasmodium that are genus or species specific. A commonly detected antigen in RDTs is PfHRP2 (Histidine-rich protein 2) [9].

The erythrocytic phase of the parasite life cycle is where clinical symptoms begin and it is the target of many antimalarials. Basically, the release of the toxic by-products that arise from the rupture of the red blood cell (RBC) membrane with merozoites into the blood stream is the reason for the high fever in malaria infections [10]. The disease is characterised mainly by fever, chills, nausea and sweating. Serious clinical manifestations result in acute renal failure, severe anemia, hemoglobinuria and comas. Moreover, P. falciparum, is referred to as the most lethal species of its kind because it can cause cerebral malaria which is a result of blocking of small blood vessels with P. falciparum-infected RBC`s [11]. Furthermore, in severe malaria cases, if left untreated, death of the infected person is almost inevitable.

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9 All Plasmodium species that cause human disease can have a recrudescence event. It is an event where asexual stages rise again in the blood stream of the host after incomplete treatment with antimalarial drugs. Relapse is a different case from recrudescence [12]. It is a special feature of Plasmodium ovale and Plasmodium malariae, where the asexual stages show up in the blood stream of the host after eliminating the parasite because they have the dormant hypnozoites in the host liver. These hypnozoites, after weeks or months of malaria elimination, form hepatic schizonts that release merozoites into the blood stream.

4. Efforts in malaria control

Malaria remains one of the 21^st century public health challenges of vector-borne diseases. The right strategy, cost, proper application and execution are key elements in a successful program to eliminate a pathogen. For the parasite to stay alive, it can only be in humans or mosquitoes. If the parasite was in the human host, it would have to be eliminated to remove the burden.

However, if the parasite is in the vector, then the vector must be controlled. According to the Centers for Disease Control and Prevention, adult male Anopheles mosquitoes do not transmit malaria because they satisfy their appetite by feeding on nectar and sugars and they live for approximately a week. Female Anopheles mosquitoes (the responsible vectors for malaria transmission) do feed on sugar sources for energy but they also require blood meals for eggs development and live up to 2 weeks. There are approximately 70 species of the Anopheles mosquitoes that are vectors for the human malaria parasites, the main being: Anopheles gambiae (known to be out-door biters) and Anopheles funestus (known to be in-door biters). Current methods of vector control include [13, 14]:

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10 1- Insecticidal-treated mosquito nets (ITN): It is a large net with pores that are small enough to not allow mosquitoes in, treated with insecticides that last for a certain period of time, and it works as a barrier between the human host and the vector. This method is cost- effective, especially for endemic areas where it has to be affordable otherwise it cannot be purchased or used, and is also very effective in blocking transmission. In fact, this application reduced malaria transmission in Sub-Saharan Africa where malaria was endemic by more than 50% [14]. However, a few shortcomings are: 1) Some adults, and especially children under five years of age, do not apply the ITNs [15, 16]; 2) Some people cannot afford them or cannot obtain them; and 3) Some mosquitoes have developed resistance to the insecticides [17, 18].

2- Indoor residual spraying: This is the best method to eliminate in-door mosquitoes. In fact, it was the principle method for the Global Malaria Eradication Campaign [19, 20]. The main deficit of this strategy is that dichlorodiphenyltrichloroethane (DDT) is environmentally unfriendly. It was used in the past and now banned in many endemic countries because of its environmental impact is [21, 22].

3- Larval source management: It is the management of mosquito breeding sites. These areas are usually small aquatic habitats that play a role in malaria transmission [23]. This strategy can block the life-cycle of the vector completely because, if these sites were controlled, there would be no new generation of mosquitoes to arise. Only a few show that this strategy contributed to reducing in-door and out-door biting and out-door breeding of the vector [24]. There are multiple ways of managing breeding sites: 1- Draining of surface water. 2- Adding predators that would live and feed on the larvae for

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11 example a fish called Gambusia affinis, a larvivorous fish. 3- The addition of an insecticide would be able to eliminate the larvae [25].

Ultimately, to treat malaria in humans, the antimalarial drugs are the best way. However, many efforts are underway to develop a protective vaccine especially for children under the age of 5 and pregnant women, who are at greater risk of infection [26].

5. Malaria vaccine

Small pox and polio have been eradicated by effective vaccines. Indeed, it would be ideal to have a malaria vaccine to add to the antimalaria arsenal [27]. Many approaches have been conducted by different groups in order to make an effective vaccine against malaria. One of these approaches was the RTS,S/AS01 vaccine which targets PfCSP (P. falciparum circumsporozoite protein), it was predicted to lead to the death of the parasite in the pre-erythrocytic stage [26, 28].

6. Hemoglobin degradation

Plasmodium parasites are dependent on the host red blood cells as a normal habitat. The asexual erythrocytic phase of the life-cycle begins with merozoites invading host RBC’s developing into rings then trophozoites then to schizonts. In order to develop and morphologically change the parasites require the products of hemoglobin degradation because it is a source of nutrition and amino acids to the parasites. Parasite invasion of host RBC’s results in the ingestion of large amounts of hemoglobin into the DV organelle. Within the DV, hemoglobin is degraded with proteolytic enzymes to globin moieties that are degraded further into smaller peptides that are important for protein synthesis [29]. The heme part is ~20mM Fe²⁺and is toxic to the parasite if not dealt with. Fe²⁺rapidly oxidizesto Fe³⁺ (also known as: ferriprotoporphyrin IX or FPIX) which

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12 is very insoluble and as such can disrupt membrane integrity and initiate reactive oxygen species that include H2O2 and OH radicals. Therefore, to avoid serious threats to its survival, the parasite converts heme into beta hematin that biomineralize to form non-toxic inert crystals known as hemozoin or malaria pigment in the DV that are visible using light microscopy [30, 31]. Once merozoites rupture from the schizonts and host RBCs, hemozoin crystals are released in the blood circulation and later pursued by macrophages for further clearing.

7. Antimalaria drugs

Many drugs have been used in the field in order to treat and eliminate malaria. The majority of these antimalarials are active against the erythrocytic stages of the parasite development. The most common group of antimalarial drugs are quinolines that include quinine (QN), quinidine, chloroquine (CQ), amodiaquine, primaquine, piperaquine, mefloquine (MQ), halofantrine (HF) and lumefantrine. Antifolates include sulphonamides, pyrimethamine and proguanil.

Hydroxynaphthaquinones includes atovaquone. Antimicrobials that include tetracycline, doxycycline and clindamycin. Lastly, artemisinin include artemisinin, dihydroartemisinin, artesunate artemether and arteether [32, 33]. Quinolines are a family of compounds that exert their effects against the asexual stages of the parasite life-cycle. These compounds are weak bases and as such accumulate in the acidic DV compartment and are suggested to interfere with hemozoin formation as observed with QN and CQ [32]. Other quinolines possess properties to eliminate liver dormant stages hypnozoites and sexual stages gametocytes, like primaquine [33- 35]. Antifolates exert their effects on the erythrocytic schizont stage. They are a class of compounds that interfere with an essential metabolic pathway that produces tetrahydrofolate that is crucial for DNA synthesis. The enzymes targeted by these compounds are dihydrofolate

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13 reductase (DHFR) and dihydropteroate synthase (DHPS) [36]. Atovaquone is a broad spectrum antiprotozoal drug that targets electron transport in the mitochondria by binding cytochrome bc1 complex and as such disrupting the membrane potential in Plasmodium parasites. This compound is always used in a synergistic combination with the antifolate proguanil [37].

Antimicrobials are inhibitors for prokaryotic microorganisms but it was shown that they do display anti-Plasmodial activity. Studies suggest that they act on the apicoplast. These compounds are slow acting and are usually used in combination with a fast acting antimalarial drug e.g. artemisinin [38]. Artemisnins are plant-derived peroxides and are the fastest acting antimalarials that kill blood stages and suppress gametocytes. Their combination with longer half-life compounds are used in the field as the first-line of defense against malaria [39].

The following section will discuss a few of the most common antimalarials in more detail.

7.1. Quinine

Quinolones are a group of compounds that contain a quinolone ring. The earliest known quinolone is an aryl-amino alcohol extracted from the bark of the cinchona tree (South America) named quinine (Qualaquin™). It was introduced to Europe in the 17^th century [28]. QN and its enantiomer quinidine are used in severe malaria cases and chloroquine-resistant malaria cases [40]. QN is believed to act on the heme-polymerization pathway, which is mainly the target of quinolone-based drugs. However, it was recently suggested that QN acts by binding a critical component of the purine salvage pathway: P. falciparum cytosolic purine nucleoside phosphorylase (PfPNP) [41]. Synthesis of QN is expensive because of its complex structure. So for now, the cinchona bark remains the main source of QN for treatment and commercial use [42].

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14

7.2. Chloroquine

This synthetic analogue of QN was synthesised in the 1930s by German chemist Hans Andersag.

It was later taken by the United States of America after World War II [43]. Quinine therapy was replaced with CQ (commercial name Resochin®) in the 1940s in efforts to eradicate malaria by the WHO [44]. Many features accompanied this drug including: 1- It was very effective. 2- It was low in toxicity. 3- It was cheap to make, which made it available in low income areas. Moreover, it was effective against all Plasmodium species that cause human disease. Moreover, the half-life of CQ is approximately 60 days [45]. For this reason it was used as a prophylactic drug [46]. It was also prescribed for pregnant women [47].

Structurally, CQ is a diprotic weak base and once protonated, it accumulates in the parasite the DV, the site of hemoglobin degradation [48, 49]. The mechanism by which CQ works is still not clear, however, it is thought that CQ acts by binding heme and preventing heme conversion into inert crystals known as hemozoins. It was believed that this would lead to the accumulation of heme monomers that later causes parasites to die [50]. Currently, CQ it is not prescribed to treat P. falciparum infected patients as the first-line of treatment and some countries withdrew the drug for a significant period of time [51]. A few years later, the malaria field saw the re- emergence of CQ-sensitive parasites. Malawi, Kenya, Cameroon and Senegal are countries that have reported a decline in CQ resistance due to the discontinuation of CQ use and/or withdrawal of CQ as the first-line of treatment [52, 53]. This pattern of re-emergence of CQS strains speculates that CQR is accompanied with a price of fitness to the parasites [54].

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15

7.3. Mefloquine and Halofantrine

In response to CQ resistance cases that affected American troops in the 1960`s, the Walter Reed Army Institute of Research in Washington DC researched drugs that were rejected as candidates after WWII [55]. This work led to the discovery of MQ and HF. MQ (Lariam®), 4- methanolquinoline, is a drug approved for clinical use by the Food and Drug Administration in 1989 [56]. MQ was used as a prophylactic drug because its half-life was 10 – 23 days. The mechanism of action of MQ is not yet understood, however like quinolone-based drugs, it is believed to act on the heme detoxification pathway [57]. MQ is now used in effective combinations with artesunate in treating uncomplicated malaria infections in adults and children [58].

HF, an aryl-amino-alcohol, is an effective antimalaria drug acting against CQR and CQS P.

falciparum. Unfortunately, due to HF`s associated cardiotoxicity, the drug was withdrawn from international guidelines in treating malaria-infected individuals [59].

7.4. Artemisinin

Chinese traditional herbal medicine treated cases of fever, then named “qinghaosu”, a sweet wormwood plant, now called Artemisia annua, in early 340 A.D. Western research confirmed artemisinin`s potent antimalarial activity in the 1990`s [59]. Artemisinin is now a major player in malaria elimination programs [60]. The best source of artemisinin is from the plant, however, this is not ideal due to the dependence of the plants on climate [61]. Unfortunately, chemical production has seen drawbacks due to the complicated structure of the compound [62].

However, genetically modified yeast has been reported to increase production of the drug [63].

Furthermore, artemisinin`s original form is not well absorbed, therefore, multiple easily absorbed

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16 derivatives have since been produced, such as artemether, artesunate and dihydroartemisinin [64].

The mode of action of artemisinin’s is not clear; some researchers suggest it acts on targets in the DV such as plasmepsin II, which is one of the enzymes involved in hemoglobin degradation [65-67]. The half-lives of artemisinin derivatives is relatively short [64, 68]. Although this is thought to reduce the likelihood of resistance, there are cases of resistance to artemisinin derivative monotherapy [69]. The WHO provided directions in 2001 on artemisinin’s drug administration, by pairing artemisinin derivatives with other effective antimalarial drugs with a different mode of action (e.g. MQ, amodiaquine, sulfadoxine-pyrimethamine, piperaquine and lumefantrine (artemether-lumefantrine is commercially known as Coartem®) in a double-barrel approach known now as Artemisinin Combination Therapy or ACT [70]. Artemisnins are administered as a 3-day regimen in combination with a longer half-life partner drug [71]. The ACT approach reduces the probability of drug resistance because the parasite has to avoid lethal effects of both agents at the same time. Unfortunately, a few cases of emerging resistance to ACTs in the Thailand-Cambodian border and in some African countries have been reported [72, 73]. Many proteins are being investigated to uncover the molecular mechanism of resistance to ART led to the discovery of mutations in k13 propeller region thought to be important in ART inactivation [74]. Be that as it may, ACT are still the first-line of treatment of uncomplicated malaria from P. falciparum in endemic regions by WHO guidelines [75-77].

8. Resistance in P. falciparum

P. falciparum is a parasite of medical importance. While the DV organelle is one target, a few other targets worthy of exploiting associated with the DV include the DV pH, redox-cycle, DV

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17 enzymes and DV integral membrane proteins. Moreover, efforts are ongoing to bring CQ back into the field. Therefore, an investigation on CQR and the possible members responsible for this process is required.

9. Chloroquine resistance

CQ was effective and efficient in curing malaria for many years [78]. Its low toxicity levels and low production cost are some of the desired features for drugs used in the field. Like many effective antimalarial drugs used against P. falciparum, resistant phenotypes emerged [79, 80]. After approximately 20 years of its usefulness in the field, cases of CQR emerged in Thailand in 1957 and by the 1970s resistance spread through the African region (sub-Saharan Africa) [7, 81].

Antimalarial drugs with similar features or superior to CQ are urgently needed. For this, understanding the molecular mechanism of CQR became a priority for many researchers in the field. CQ is thought to act on the heme biocrystallisation pathway [31, 82]. Verapamil, an L-type calcium channel blocker, also known as an inhibitor to P-glycoprotein (or P-gp, an ABC transporter associated with drug resistance in cancer cells) was shown to reverse CQR in CQ resistant strains [83]. ABC transporters are known for their ability to export many anti-cancer drugs. This suggested that CQR could be due to a homolog of MDR in P. falciparum which was proven otherwise with a genetic-cross between Dd2 and HB3 [84, 85].

10. Transporters implicated in drug resistance 10.1. Pfkelch13

Artemisinin combination therapies (ACT) are the first-line of treatment recommended by the WHO against P. falciparum parasites [86]. Current successes with ACT is now threatened by the emergence of resistance seen in the malaria field [87]. Resistance emerged in western Cambodia

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18 and now has spread to Southeast Asia and South China [69, 88-90]. Using whole genome sequencing of an ART sensitive parasite line pressured for approximately 5 years with ART uncovered a gene designated PF3D7_1343700 (k13) also known as Kelch protein or Pfkelch13 and mutations in this intron-less gene were linked to ART resistance [74]. There are over a hundred different k13 mutations but the most frequent are R539T, C580Y and Y493Y [91-93].

Pfkelch13 is found to localise in vesicles that reside close to the parasite produced erythrocyte- cytosol containing structures known as cytostomes (cell mouth). Using human KEAP1 as a template, the structure of Pfkelch13 by homology modeling is composed of three functional domains: a Plasmodium-specific localisation sequence on the N-terminus, BTB/POZ domain that is known to facilitate ubiquitin-mediated degradation and a six blade carboxyl-terminal Kelch propeller repeat domain predicted to function as a scaffold for protein-protein interactions [74, 94, 95]. Based on the Kelch/BTB/POZ family of adaptors, the protein is speculated to function as a regulatory protein [96, 97]. It was shown that Pfkelch13 proteins in such compartments were required for hemoglobin endocytotic uptake [94]. Interestingly, parasites with inactivated PfKelch13 or resistance-conferring mutation of PfKelch13 showed lower hemoglobin uptake and enhanced ring stage resistance to ART when exposed to high concentrations of ART [94, 95]. A model proposed that when the ability of Pfkelch13 to assist in hemoglobin engulfment was impaired due to mutations in the propeller domain resulted in less hemoglobin breakdown and as such less heme is available for ART activation [98]. This process slows the parasites growth because of the reduced amino acid pools derived from hemoglobin degradation that is needed for synthesis. This in turn leads to cellular stress relieved by unfolded protein responses [98-100].

ART inhibition of hemoglobin engulfment suggests a possible target to explore for future

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19 antimalarials [101]. Resistance to ART is not limited to the Pfkelch13 domain, other proteins have are being investigated in this spectrum including PfMDR1, AP-2μ, coronin, falcipain 2, UBP1 and PfPI3K [102-111].

10.2. ABC transporters in Plasmodium spp.

There are 48 ABC transporter genes in humans, divided into 7 subfamilies from A to G [112]. By contrast, the Plasmodium species encode only 16 ABC transporters, divided into 6 subfamilies: B, C, E, F, G and I [112]. These proteins require adenosine-tri-phosphate (ATP) as fuel for their transport functions. ABC transporters are found in humans as well as many parasites [113]. They are composed of two transmembrane domains with 6 alpha-helices each (the number of alpha helical domains can vary between different members of the ABC-transporters) and two nucleotide binding domains (NBD) in the cytosol [114]. Each NBD contains Walker A and Walker B consensus sequences separated by approximately 90 - 120 amino acids. The NBDs share high sequence homology between the different members of the ABC transporters. In contrast, the transmembrane domains share little or no sequence homology [114]. Both P-gp and MRP have been shown to transport a large array of substrates and have been implicated in drug resistance in tumor cells [115, 116]. In addition, the overexpression of P-gp and/or MRP in tumor cells results in reduced intracellular drug concentration through active drug efflux [115, 116].

Homologues of P-gp and MRP have been described in P. falciparum (e.g., PfMDR1, PfMRP1 and PfMDR2 [117, 118]).

10.2.1. PfMDR1

Early evidence suggested that the chloroquine-heme complex formation, in the DV acidic organelle in the parasite, was responsible for the anti-Plasmodial activity of CQ [119, 120]. It was

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20 also observed that there was less CQ accumulating in CQR strains [121, 122]. Moreover, reversal of CQ resistance with VP and reduced accumulation of CQ in resistant parasites resembled behavior of multidrug resistance in mammalian cells similar to P-gp [84, 121, 123, 124]. This striking resemblance to P-gp prompted the discovery of transporter homologs of mammalian drug resistance in P. falciparum genes [125, 126]. The P. falciparum genome encodes 7 members of the ABCB subfamily [117]. P. falciparum multidrug resistance protein 1 or PfMDR1 (also known as P-glycoprotein homolog 1 or P-gh1) gene is located on chromosome 5 and is intronless. It encodes a single protein composed of 1419 amino acids and a molecular mass of 162.25kDa and is expressed throughout the asexual life stages of the parasite localising to the DV membrane [125, 127-129]. Like a typical ABC transporter, the protein structure is predicted to be of two domains consisting of six predicted transmembrane alpha helixes with two NBDs (the NBDs are on the cytosolic side of the DV membrane [129, 130]) joined together with a linker domain [128, 131]. PfMDR1 is expressed in CQS strains and overexpressed in some CQR strains; additionally, point mutations in PfMDR1 of CQR strains have been associated with chloroquine resistance [125, 132]. Another study showed that mutations in PfMDR1 can modulate the levels of CQR in cells that possess mutant-PfCRT but cannot confer resistance to CQ by themselves [133].

Selecting mutants pressured in high concentrations of CQ caused de-amplification of PfMDR1 and increased sensitivity to MQ [134]. However, selecting mutants pressured in high concentrations of MQ caused gene amplification of PfMDR1 which correlates with the increase of PfMDR1 transcripts and protein levels in MQ resistant strains [135, 136]. Indeed, the key determinant of MQ, QN and HF is the amplification of PfMDR1 [135]. Five polymorphisms in PfMDR1 appear to contribute to CQR N86Y, S1034C, N1042D, D1246Y and Y184F [132, 133, 137].

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21 Moreover, heterologous expression of wild-type PfMDR1 in CHO cells increased CQ accumulation in the cells making them more sensitive to CQ. This lead to the hypothesis that PfMDR1 may function as an importer of CQ into the DV of the parasite [138]. However, when mutant PfMDR1 containing mutations S1034C and N1042D that were thought to be associated with CQR did not make the cells more sensitive to CQ which suggest that some mutations alter the import of CQ via PfMDR1 [138]. Similarly, expression of wild-type PfMDR1 in frog system Xenopus laevis oocytes showed increased sensitivity to CQ and less accumulation of CQ when mutant form was expressed and this suggests that resistance to CQ conferred by PfMDR1 would be reduced or abolished import of CQ into the DV [139, 140]. The normal substrate of PfMDR1 is un-known but it has been shown to import a variety of synthetic compounds such as Fluo-4, Fluo-4AM, CQ, AQ, QN, MQ, HF and ART [129, 133, 140-142]. Moreover, it was suspected that PfMDR1 is involved in parasite sensitivity to ACT partner drugs and artemisinin derivatives [143-145]. In-vitro selection of parasites incubated with artelinic acid and artemisinin resulted in parasites less sensitive to ART in addition to MQ, QN, HF and Lumefantrine [102]. Recently, a study involving 37 malaria positive patients in Nigeria detected rare mutations in PfMDR1 speculated to be involved in resistance to antimalarials used in the field (N504K, N649D, F938Y and S967N) that demonstrates the everlasting resistance that comes with treating with antimalaria drugs [146].

10.2.2. PfMRP1

Other members of the ABC transporters superfamily implicated in drug resistance in P.

falciparum are from the ABCC sub family; members included: PfMRP1 and PfMRP2 [147].

Topology of PfMRP1 suggests a composition of 12 membrane-spanning alpha helices with 2 NBDs extending into the cytosol [148]. These proteins are expressed on the plasma membrane and

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22 membrane-bound vesicles throughout the asexual life stages of the parasite, but not on the DV membrane such as PfMDR1. Some PfMRP1 single nucleotide polymorphisms were linked to decreased sensitivity of the parasite to CQ and QN [117, 149]. Indeed, mammalian MRP1 was reported to transport the antioxidant GSH as well as leukotriene C4. Similarly, studies suggested that PfMRP1 actively transports GSH in addition to GSH-conjugates, CQ, QN and ART in an efflux manner [147]. Moreover, studies have shown a defect in the fitness of the parasite when the function of the gene encoding PfMRP1 was disrupted in-vivo, showing more GSH accumulation as well as the inability of the parasites to survive beyond 5% parasite density in addition to increased sensitivity to CQ, QN and ART [150]. All of this could be due to the possible role of PfMRP1 to export toxic waste and support a healthy parasite life-cycle.

Even though PfMRP1 influences CQR to a certain degree, it was not expressed on the DV.

Therefore, it was not the target of verapamil that had the major influence on CQR. Moreover, in the case of human MRP1 (ABCC1), verapamil increases the transport activity of MRP1 for GSH [151] which can result in increased sensitivity of tumor cells to oxidative stress [152].

10.2.3. PfABCG

Unlike many ABC transporters that are usually composed of approximately 12 transmembrane domains with two NDB`s in a single polypeptide chain, the members of the G subfamily consist of 6 membrane spanning alpha helices with a single NBD, but they function as homo- or hetero- dimers [117]. Interestingly, while mammalian cells encodes 5 members of the G subfamily, the parasite encodes only one ABCG protein, known as PfABCG [117]. Moreover, a protein BLAST with a local alignment suggests that PfABCG shares sequence Identity with human ABCG1 and human ABCG2. It has been shown that PfABCG is expressed in all parasite blood stages (rings,

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23 trophozoites, schizonts and gametocytes)[153]. Interestingly, it has also been shown that PfABCG localises to the plasma membrane as well as to a special unidentified organelle in the parasite [153]. Furthermore, human ABCG1 was shown to transport cholesterol and other sterols, while human ABCG2, known as BCRP because of its involvement in breast cancer drug resistance, was shown to transport many anti-cancer drugs, as well as GSH, uric acid and heme [154]. However, the normal function of PfABCG is still a matter of speculation but some studies suggest that it transports ketotifen (an anti-histamine)[155]. The predicted molecular weight of PfABCG is approximately 75kDa.

10.3. PfCRT

The evidence that was pointing to the primary determinant of CQ resistance in P. falciparum was that resistance can be reversed significantly in the presence of verapamil in CQR strains. Later, a genetic-cross between Dd2 (CQR) and HB3 (CQS) parasites showed that there was no link between mdr-like genes and CQR. Instead, the examination of chromosome 7 of the same genetic cross revealed a unique gene sequence of 13 exons that encodes a highly polymorphic transporter, then named the P. falciparum CQ resistance transporter, abbreviated PfCRT [156, 157].

PfCRT is an integral membrane protein localising to the DV membrane of P. falciparum [156]. The recently resolved structure of PfCRT-CQR-7G8 strain (UNIPROT W7FI62) at 3.2Å resolution shows that PfCRT is composed of ten membrane-spanning alpha-helices with the N- and C- termini extending into the cytosol [158]. Indeed, it was shown that the key determinant for CQR is a mutation in TMD1 at position 76 [85, 159]. Moreover, the amino acid substitution of the positively charged lysine (K) with the non-charged threonine (K76T) is a conserved mutation in

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24 CQR strains [85, 159]. To date, there are three phosphorylation sites reported in PfCRT, all in the cytosolic domains: N-terminal Ser33 and C-terminal Ser411 and Thr416. Moreover, phosphorylation of Thr416 was demonstrated to be a possible deciding signal for trafficking of PfCRT to its location on the DV [160]. A later report demonstrated with an inducible expression of PfCRT-GFP fusion protein, where it showed expression starts at the ring stage in pre-DV compartments, then the expression peaks at the trophozoite stage at mature DV stage [161].

Bioinformatics analyses suggest PfCRT is a member of the drug/metabolite transporter superfamily of electrochemical potential-driven transporters [162, 163].

10.3.1. PfCRT polymorphisms and chloroquine resistance

PfCRT has been shown to encode 4 to 10 mutated residues in addition to the K76T mutation in CQR strains, with a few exceptions to some strains that have a K76A variant or K76I or K76N that are both laboratory strains that have undergone drug pressure with CQ [156, 164, 165]. To date, there are 32 polymorphisms associated with this protein. The number of mutations PfCRT can possess and how it acquires them is unknown. However, it could be due to different drug pressure selections depending on the geographical region of the parasite. Moreover, the K76T mutation is the most conserved mutation in CQR strains. Interestingly, restoring K76T mutation back to lysine reversed CQ resistance demonstrating that the latter amino acid at position 76 in PfCRT is a key determinant for CQ resistance [159]. Heme (Fe²⁺) is a by-product of hemoglobin digestion that is rapidly oxidised to form insoluble Fe³⁺(ferriprotoporphyrin IX or FPIX), which is lethal because it can disrupt membrane function and release reactive oxygen species [166]. To survive in such conditions, the parasite bio-crystallises heme into inert hemozoin crystals which are visible using light microscopy [29-31, 167]. The mechanism of CQ resistance is still not entirely

Biochemical Characterisation of The Plasmodium falciparum. Chloroquine Resistance Transporter